Multifunctional aromatic compounds, which comprise an aromatic ring and two or more functional groups, have potential utility in a wide variety of industrial applications. Examples include para-hydroxycinnamic acid (pHCA), cinnamic acid (CA), and para-hydroxystyrene (pHS). These aromatic compounds have application in monomers for the production of liquid crystalline polymers, and in the production of resins, elastomers, coatings, adhesives, automotive finishes and inks.
Chemical synthetic methods for producing these aromatic compounds are known. However, these chemical methods are expensive due to the high cost of the starting materials and the extensive product purification required. Moreover, these methods generate large amounts of unwanted byproducts. Consequently, biological production methods for these aromatic compounds have been developed. For example, Gatenby et al. in U.S. Pat. No. 6,368,837 describe several methods for producing pHCA from glucose using recombinant microorganisms. A biological process for the production of pHS from a simple carbon source such as glucose is described by Ben-Bassat et al. in co pending U.S. Patent Application No. 60/383,450. Additionally, Qi et al. in co pending U.S. patent application Ser. No. 10/138,970 describe methods for producing CA and pHCA using recombinant microorganisms. However, a problem encountered with the biological production of these aromatic compounds is end-product inhibition, which limits product yield. Specifically, the rate of production of the product by the microorganism decreases as the concentration of the product increases. Additionally, the microorganism becomes inactivated by the product when a certain critical concentration is reached in the fermentation medium.
One approach to mitigate end-product inhibition is to use two-phase extractive fermentation, in which the product is extracted into an immiscible organic phase during the fermentation so that it never reaches an inhibitory or critical concentration. In this way, the microorganism can function at a high rate of production over an extended period of time.
The principle of two-phase extractive fermentation was first described by Dreyfus in U.S. Pat. No. 2,053,770. In that disclosure, the extractive fermentation of alcohol and acetone/butanol using organic solvents such as isoamyl alcohol is described. Kollerup et al. in U.S. Pat. No. 4,865,973 and European Patent No. 0216221 describe a process for the two-phase extractive fermentation of ethanol, acetone/butanol, penicillin, citric acid, and polysaccharides. In those disclosures a variety of solvents are taught, including: double bond unsaturated aliphatic alcohols having 12 or more carbon atoms; saturated branched chain aliphatic alcohols having 14 or more carbon atoms or mixtures thereof; double bond unsaturated aliphatic acids having 12 or more carbon atoms; aliphatic and aromatic mono-, di- or tri-esters having 12 or more carbon atoms, other than dibutyl phthalate; aliphatic noncyclic ketones and aliphatic aldehydes having 12 or more carbon atoms; and mixtures thereof.
There are other examples of two-phase extractive fermentation in the art. For example, see Jones et al., Biotechnol. Lett. 15:871–876 (1993); Daugulis et al., Biotechnol. Lett. 16:637–642 (1994); Lewis et al., Biotechnol. Prog. 8:104–110 (1992); and Barton et al., Appl. Microbiol. Biotechnol. 36:632–639 (1992). Lee et al. (Enzyme Microb. Technol. 23:261–266 (1998)) describe the production of 4-vinylguaiacol (4-hydroxy-3-methoxystyrene), a derivative of pHS, via the decarboxylation of ferulic acid by resting cells of Bacillus pumilus using a two-phase, biocatalytic process. Several solvents were evaluated, including chloroform, methylene chloride, ethylacetate, ethyl ether, petroleum ether, cyclohexane, and C5–C8 alkanes. Hexane was selected as the preferred solvent. All of these solvents are toxic to microorganisms and/or are hazardous, and therefore, are unsuitable for the commercial scale production of multifunctional aromatic compounds via fermentation.
As indicated by the above, although methods have been developed for two-phase extraction of fermentation products, there have been no reports of the use of extractive fermentation in the production and recovery of pHCA, CA, or pHS. This may be in part because the development of novel two-phase extractive fermentation systems is a difficult and arduous task for one skilled in the art because of the unpredictable nature of these systems. For example, the selection of the solvent is very critical and the optimal solvent must be determined for each product/microorganism combination. The solvent must meet the following requirements for use in a commercial two-phase extractive fermentation process: low solubility in water, nontoxic to the producing microorganism, large partition coefficient for the product, low partition coefficient for nutrients, high selectivity, low emulsion forming tendency, high chemical and thermal stability, nonbiodegradability, nonhazardous, and low cost (Bruce et al., Biotechnol. Prog. 7:116–124 (1991)). Methods for predicting the biocompatibility of organic solvents (Bruce et al., Supra) have been suggested, however those methods address only one of the parameters needed for the development of a two-phase extractive solvent system. Moreover, those methods only serve as a guide for the selection of non-toxic solvents. The actual toxicity of the solvent can only be determined by experimental testing.
Toxicity testing of the solvent system is a particular problem and solvent toxicity is difficult to predict (Eiteman et al. Appl. Microbiol. Biotechnol. 30:614–618 (1989)). Playne et al. (Biotechnol. Bioeng. 25:1251–1265 (1983)) report diisoamyl ether (diisopentyl ether), is nontoxic toward anaerobic bacteria, as determined using flask tests. Staley et al. in WO 01/98521 describe a method for extracting carboxylic acids from fermentation broth using 2-decanone. Collins et al. (Biotechnol. Bioeng. 55:155–162 (1997)) and Biotechnol. Techniques 10:643–648 (1996)) describe the biodegradation of phenol by Pseudomonas putida in two-phase bioreactors using 2-undecanone as solvent. In that disclosure the organic solvent was used to control delivery of a toxic substrate, not to extract the product of a fermentation. Eiteman et al., supra, describe experimental testing of 24 solvents for two-phase extractive fermentation to produce 2,3-butanediol using Klebsiella oxytoca. 
None of the above described solvents have been reported for the extraction of multifunctional aromatic compounds such as pHCA, CA, and pHS, and clearly a need exists for fermentative extraction of these compounds.
Applicants have met the stated need by providing a method for the production of multifunctional aromatic compounds, such as pHCA, CA, and pHS, in high yield using two-phase extractive fermentation with a novel group of extractive solvents.